Biomarkers and personalised medicine in rheumatoid arthritis: a proposal for interactions between academia, industry and regulatory bodies.

Rheumatoid arthritis (RA) is one of the most appropriate conditions for the application of personalised medicine as a high degree of heterogeneity has been recognised, which remains to be explained. Such heterogeneity is also reflected in the large number of treatment targets and options. A growing number of biologics as well as small molecules are already in use and there are promising new drugs in development. In order to make the best use of treatment options, both targeted and non-targeted biomarkers have to be identified and validated. To this aim, new rules are needed for the interaction between academia and industry under regulatory control. Setting up multi-centre biosample collections with clear definition of access, organising early, possibly non-committing discussions with regulatory authorities, and defining a clear route for the validation, qualification and registration of the biomarker-drug combination are some of the more critical areas where effective collaboration between the drug industry, academia and regulators is needed.

Liposome-incorporated dexamethasone palmitate inhibits in-vitro lymphocyte response to mitogen.

The use of liposomes for the pulmonary delivery of corticosteroid is an area that is under active investigation. We have recently developed a novel liposomal corticosteroid preparation based on the incorporation of dexamethasone palmitate (DMP) within the bilayer of small unilamellar vesicles (SUVs) made of egg yolk phosphatidylcholine (EPC) and cholesterol; molar ratio EPCC:cholesterol: DMP, 4:3:0.3. In the present study, the biological activity of DMP-SUVs was evaluated using the lymphocyte transformation test with peripheral blood mononuclear cells (PBMCs) and a gamma-interferon production assay. Results showed that DMP-SUVs (but not empty SUVs) inhibited [3H]thymidine uptake and gamma-interferon production by concanavalin A-stimulated PBMCs by 94 and 96%, respectively, at a concentration corresponding to 10(-6) M dexamethasone. The inhibition by DMP-SUVs was found to require a 24-h pre-incubation with unstimulated PBMCs, suggesting that interaction of SUVs with lymphocytes may be altered by mitogen stimulation. We conclude that our DMP liposomal preparation is biologically active and may be considered a promising alternative to conventional local glucocorticoid therapy.

A method to assess the lysosomal residence of proteins in cultured cells.

Analytical subcellular fractionation is playing an increasingly important role in proteomic studies to identify and validate components of cellular organelles. For lysosomes, definitive studies in this area have been restricted to rodent tissues due to technical constraints. Our goal was to design a quantitative assay that would allow clear demonstration of lysosomal localization in cultured human cells. We found that culturing HepG2 (human hepatocellular carcinoma) cells in progesterone-containing medium elicited an extensive shift in the buoyant density of lysosomes as measured by isopycnic centrifugation in sucrose density gradients. The density of other organelles remained essentially unchanged; thus, this shift represents a specific test for lysosomal localization. Progesterone treatment of a variety of other cultured cells also elicited a shift in lysosome density. This approach should represent a valuable tool for identification and validation of both luminal and membrane lysosomal proteins.

Increased renal vascular reactivity to ANG II after unilateral nephrectomy in the rat involves 20-HETE.

This study examined the role of intrarenal ANG II in the renal vascular reactivity changes occurring in the remaining kidney undergoing adaptation following contralateral nephrectomy. Renal blood flow responses to intrarenal injections of ANG II (0.25 to 5 ng) were measured in anesthetized euvolemic male Wistar rats 1, 4, 12, and 24 wk after uninephrectomy (UNX) or sham procedure (SHAM). At week 4, renal vasoconstriction induced by 2 ng ANG II was greater in UNX (69 +/- 5%) than in SHAM rats (50 +/- 3%; P < 0.01). This response was inhibited, by 50 and 66%, and by 20 and 25%, in SHAM and UNX rats, after combined injections of ANG II and losartan, or PD-123319 (P < 0.05), respectively. Characteristics of ANG II receptor binding in isolated preglomerular resistance vessels were similar in the two groups. After prostanoid inhibition with indomethacin, renal vasoconstriction was enhanced by 42 +/- 8% (P < 0.05), only in SHAM rats, whereas after 20-HETE inhibition with HET0016, it was reduced by 53 +/- 16% (P < 0.05), only in UNX rats. These differences vanished after concomitant prostanoid and 20-HETE inhibition in the two groups. After UNX, renal cortical protein expression of cytochrome P-450 2c23 isoform (CYP2c23) and cyclooxygenase-1 (COX-1) was unaltered, but it was decreased for CYP4a and increased for COX-2. In conclusion, renal vascular reactivity to ANG II was significantly increased in the postuninephrectomy adapted kidney, independently of protein expression, but presumably involving interactions between 20-HETE and COX in the renal microvasculature and changes in the paracrine activity of ANG II and 20-HETE.

Water permeability of apical and basolateral cell membranes of rat inner medullary collecting duct.

To quantify the pathways for water permeation through the kidney medulla, knowledge of the water permeability (Posmol) of individual cell membranes in inner medullary collecting duct (IMCD) is required. Therefore IMCD segments from the inner two thirds of inner medulla of Sprague-Dawley rats were perfused in vitro using a setup devised for rapid bath and luminal fluid exchanges (half time, t1/2, of 55 and 41 ms). Differential interference contrast microscopy, coupled to video recording, was used to measure volume and approximate surface areas of single cells. Volume and volume-to-surface area ratio of IMCD cells were strongly correlated with their position along the inner medullary axis. Transmembrane water flow (Jv) was measured in response to a variety of osmotic gradients (delta II) presented on either basolateral or luminal side of the cells. The linear relation between Jv and delta II yielded the cell membrane Posmol, which was then corrected for membrane infoldings. Basolateral membrane Posmol was 126 +/- 3 microns/s. Apical membrane Posmol rose from a basal value of 26 +/- 3 microns/s to 99 +/- 5 microns/s in presence of antidiuretic hormone (ADH). Because of amplification of basolateral membrane, the ADH-stimulated apical membrane remained rate-limiting for transcellular osmotic water flow, and the IMCD cell did not swell significantly. Calculated transcellular Posmol, expressed in terms of smooth luminal surface, was 64 microns/s without ADH and 207 microns/s with ADH. IMCD cells in anisosmotic media displayed almost complete volume regulatory decrease but only partial volume regulatory increase.

Adaptation of inner medullary collecting duct to dehydration involves a paracellular pathway.

Prolonged fluid restriction in rats is accompanied by functional modifications of the terminal part of the inner medullary collecting duct (IMCD) revealed by a sustained increase in arginine vasopressin (AVP)-independent transepithelial osmotic water permeability (PTE) in vitro. The cellular basis of this adaptation was explored in isolated and perfused terminal IMCDs of Sprague-Dawley rats using video and fluorescence microscopy. Basolateral membrane osmotic water permeability (Posm), transcellular Posm, and PTE were measured in quick sequence in every tubule. They were expressed per unit area of basolateral membrane corrected for infoldings, based on previous stereological studies and assuming no major change in membrane surface area between hydrated and dehydrated animals. Compared with IMCDs of rats with a high water intake, IMCDs of rats deprived of fluid for 36 h displayed a significantly higher basal PTE (24.9 +/- 5.1 vs. 6.1 +/- 0.6 microns/s), a similar basolateral Posm, and a higher transcellular Posm, implying a higher permeability of the apical membrane, despite the absence of exogenous AVP. However, when IMCDs of thirsted rats were exposed to AVP in vitro, their transcellular Posm (36.0 +/- 2.4 microns/s) was significantly smaller than their PTE determined simultaneously (51.8 +/- 7.1 microns/s), suggesting that part of the water flow may follow a paracellular route. A change in paracellular pathways was supported by higher apparent permeabilities to [14C]sucrose (0.85 +/- 0.27 vs. 0.28 +/- 0.04 x 10(-5) cm/s) and to [methoxy-3H]inulin (0.25 +/- 0.04 vs. 0.14 +/- 0.03 x 10(-5) cm/s) in IMCDs of thirsted rats. The nonelectrolyte permeabilities were affected neither by AVP nor by urea-rich bathing solutions. We conclude that in vivo factors related to dehydration produce a conditioning effect on terminal IMCD, which includes stabilization of the apical membrane in a state of high Posm and opening up of paracellular pathways revealed by a higher permeability to water and nonelectrolytes. The role of these adaptive phenomena remains unclear but may pertain to the sudden transitions between antidiuresis and diuresis.

Drugs activating G proteins disturb cycling of ADH-dependent water channels in toad urinary bladder.

In the toad urinary bladder, antidiuretic hormone (ADH)-mediated changes in water permeability depend on exocytic insertion and endocytic retrieval of water channels into and from the apical membrane, respectively. Because GTP-binding proteins (G proteins) are well-recognized regulators of vesicular trafficking throughout the cell, we tested the hypothesis that drugs interfering with G protein would modify the hydrosmotic response to ADH and the ADH-regulated formation of endosomes, as assessed by luminal incorporation of a fluid-phase marker [fluorescein isothiocyanate (FITC)-dextran, 70 kDa]. Mastoparan (4 microM) and compound 48/80 (poly-p-methoxyphenylethylmethylamine; 50 micrograms/ml), added to the luminal side of the toad urinary bladder, as well as AlF3 added to the serosal side (400 microM), inhibited ADH- and 8-bromoadenosine 3',5'-cyclic monophosphate-induced transepithelial water flow by > 50% and simultaneously enhanced cellular incorporation of FITC-dextran by > 200%. The pattern of FITC-dextran uptake observed using fluorescence microscopy both in scraped cells and in the intact bladder was granular, suggesting fluid-phase endocytosis. Mastoparan and AlF3, which are both probes of G proteins, increased FITC-dextran uptake only in the presence of ADH and a transepithelial osmotic gradient, i.e., under conditions where water channel-carrying endosomes presumably cycle. Therefore, we suggest that the ADH-dependent cycling of water channels could be controlled by one or more G proteins associated with the apical membrane and/or the water channel-carrying vesicles.

Flow rate measurements in isolated perfused kidney tubules by fluorescence photobleaching recovery.

We have developed a new application of the fluorescence photobleaching recovery (FPR) technique for instantaneous measurement of volume flow rates at any axial position along isolated perfused kidney tubules. The method requires fast data acquisition of emitted fluorescence through a photomultiplier (time resolution, 0.5 ms) coupled with differential interference contrast microscopy to measure luminal diameters accurately. While the tubule is perfused in vitro with an impermeant fluorophore (fluorescein sulfonate), a 20-ms bleach pulse reduces the fluorescence in the observation region by 20-25%. Fluorescence recovery is a direct function of perfusate velocity; diffusion plays no significant role in the early phase of recovery. A fluid dynamics approach to data analysis shows that fractional recovery increases linearly with time until t = L/2vm, where L is the length of the observation window and vm is the mean axial velocity. Practically, a linear regression analysis of the early recovery phase allows measurement of vm of up to 0.14 cm/s, i.e., a 40-nl/min flow rate in a 25-microns-diameter tubule. Calibration experiments in small glass tubes perfused at predetermined flow rates demonstrated good accuracy (within 10%) and reproducibility (coefficient of variation, 8.7%). In rat inner medullary collecting ducts microperfused at 4-40 nl/min, the correlation with a standard fluid collection method was excellent (r2 greater than 0.97). The method should also be suitable for the direct measurement of fluid flow rate in kidney tubules or blood vessels microperfused in vivo.

Treatment of hyponatremic cirrhosis with ascites resistant to diuretics by urea.

We have studied the efficacy of urea in the treatment of hyponatremia and hydrosaline retention in cirrhotic patients with ascites resistant to diuretics. In 5 patients with hyponatremia and ascites resistant to a major diuretic treatment (200-400 mg spironolactone combined with 40-160 mg furosemide/day for 4 of them), urea intake (30-90 g/day) induced the following changes: the daily weight changed from a gain of 0.01 +/- 0.06 kg/day to a loss of 1.03 +/- 0.12 kg/day (p less than 0.001) (mean +/- SEM), serum sodium concentration rose from 128 +/- 1.3 to 133 +/- 1.4 mmol/l (p less than 0.01), sodium output increased from 24 +/- 4 to 82.5 +/- 11 mmol/day, diuresis increased from 1.05 +/- 0.10 to 2.24 +/- 0.24 liters/day (p less than 0.01). Despite an important weight loss, the creatinine clearance did not change significantly (53.6 +/- 4.5 ml/min before and 70.0 +/- 8.2 ml/min during urea). In patients responding to classical diuretics, urea as a monotherapy was less effective. From the 6 patients with resistant ascites, only 1 developed prerenal uremia after urea treatment. In order to enhance urea efficacy, it is important to take it together with a long-loop diuretic. Intermittent urea intake seemed to be useful in cirrhotic patients with hyponatremia associated with ascites resistant to diuretics and with low or normal blood urea concentrations.

Continuous ambulatory peritoneal dialysis vs haemodialysis: a lesser risk of amyloidosis?

We compared plasma beta-2-microglobulin (beta 2M) at a 1-year interval in 25 CAPD patients and 25 patients haemodialysed with cuprophane membranes and matched for residual renal function and duration of renal replacement therapy. Plasma beta 2M remained lower in CAPD patients throughout the study, and increased significantly with time both in CAPD and haemodialysis patients, as renal function decreased. In both groups, plasma beta 2M was negatively correlated with residual creatinine clearance, the influence of the latter being much greater in haemodialysis, as demonstrated by comparison of the regression lines. In haemodialysis, but not in CAPD, plasma beta 2M also correlated with time on dialysis. In CAPD patients, the daily peritoneal output averaged 38 mg (range 16-59 mg), and was directly correlated with plasma beta 2M. CAPD thus allows a significant peritoneal removal of beta 2M, which progressively takes over from the declining renal function, resulting in lower plasma beta 2M than in matched haemodialysis patients. However, the peritoneal removal of beta 2M remains insufficient and values increase with time as renal function declines. Thus, if beta 2M amyloidosis is related to raised plasma levels, the risk of beta 2M amyloidosis in CAPD should simply be delayed as compared to haemodialysis.

Comparison of elastase-1 with amylase, lipase, and trypsin-like immunoreactivity in the diagnosis of acute pancreatitis.

Serum elastase-1, amylase, lipase, and trypsin-like immunoreactivity were measured in a group of 17 consecutive patients with acute pancreatitis. When assayed within 24 h of the onset of symptoms, all enzymes were found to be elevated, thus showing similar sensitivity. Elastase-1 did not improve the diagnostic score of the other enzymes studied. Owing to their much quicker and less expensive determinations, amylase and lipase should be considered the best initial markers of pancreatic injury. However, during the course of pancreatitis, amylase and in a lesser degree lipase returned to normal in more cases than elastase or trypsin; both were still elevated in 90% of the patients 10 days after the onset of the symptoms. Thus, trypsin and/or elastase-1 should be reserved for cases of doubtful or delayed diagnosis. The specificity and the positive predictive value of these enzymes need to be evaluated.